Amplifying rare cell surface markers

ABSTRACT

This invention relates generally to a microfluidic device for encapsulation, incubation, and analysis of cell surface markers or secreted molecules from a single cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/982,458, filed on Dec. 29, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/583,478, filed on Sep. 7, 2012, which is a U.S.National Phase Application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2011/027926, filed on Mar. 10, 2011, which claimsthe benefit of U.S. Provisional Patent Application No. 61/313,667, filedon Mar. 12, 2010, the contents of which are hereby incorporated byreference in their entireties.

TECHNICAL FIELD

The invention relates to systems and methods for detecting moleculessecreted from a single cell and rare cell surface markers, inparticular, systems and methods using rolling circle amplification withmicrofluidic devices for encapsulation, incubation, and analysis of cellsurface markers or secreted molecules from a single cell.

BACKGROUND

The ability to gather statistical information over large populations ofcells using flow cytometry of cells labeled with fluorescent probes hascontributed tremendously to the diagnostics and study of cellularfunction. In a flow cytometer or a fluorescence-activated cell sorter(FACS), cells are interrogated by flowing them past a detector in acontinuous stream of buffer and sorted by molecules either confinedwithin the cells or bound to the cell surface, rather than by propertiesof the molecules the cells secrete (Köster et al., Lab on a Chip 8,1110-1115 (2008); Herzenberg et al., Clin. Chem. 48, 1819-1827 (2002);Carroll and Al-Rubeai, Expert Opin. Biol. Ther. 4:1821-1829 (2004)). Lowexpression of a target molecule can lead to an insufficient orlow-intensity detection signal. Further, to sort cells based on secretedmolecules, additional tests are usually performed by enzyme-linkedimmunosorbent assay (ELISA) or comparable methods to verify secretion ofa particular molecule of interest from a population of cells rather thanfrom an individual cell. It is also challenging to determinetime-dependent variability of secretion from single cells by currentmethods.

SUMMARY

The present invention is based, at least in part, on systems and methodsfor amplifying rare cell surface markers, in particular, systems andmethod using rolling circle amplification with microfluidic devices forencapsulation, incubation, and analysis of cell surface markers orsecreted molecules from a single cell. The methods and systems describedbelow can provide high efficiency techniques for specific detection andamplification of cell specific surface markers. The identification ofcell surface antigens can be critical to the development of newdiagnostic and therapeutic modalities for the management of diseasessuch as, for example, cancer.

The systems and methods disclosed label cell surface specific markersusing rolling circle amplification technique (RCA). RCA is a simpleamplification method with high sensitivity and specificity owing to theDNA stringent strand matching requirement and its high signalamplification efficiency (see e.g., Konry et al., Analytical Chemistry81(14), 5777-5782 (2009); Demidov, Expert Rev. Mol. Diagn., 2(6), 89-95,(2002); and Landegren et al., Comp. Funct. Genomics, 4, 525-530,(2003)). RCA technology detects and measures proteins as well as nucleicacids with unprecedented sensitivity and expanded multiplexingcapabilities. A cell specific surface marker is a molecule usually foundon the plasma membrane of a specific cell type or limited number of celltypes. These markers can be extremely useful to distinguish diseasedcells or tissues from those in normal state. The presented technique canbe applied to identify, detect, and quantify specific types of cells incomplex multi-cellular systems fixed on a glass slide, biochip platformor in flow cytometry analysis.

In one aspect, methods of for detecting cell-surface expression orsecretion of a protein or peptide by a single cell include: providing asample comprising a population of cells and reagents sufficient fordetection of a protein or peptide; dividing the sample into subsamples,such that each subsample contains at most one of the population of cellsand each subsample is encompassed a hydrophobic fluid; maintaining thesubsamples under conditions sufficient to allow detection of the proteinor peptide; and detecting the protein or peptide; thereby detectingexpression of the protein or peptide by a single cell. Embodiments caninclude one or more of the following features.

In some embodiments, the protein or peptide is expressed on the surfaceof the cell or secreted from the cell.

In some embodiments, the reagents comprises rolling circle amplificationreagents, e.g., phi29 DNA polymerase, and circular DNA template.

In some embodiments, the reagents comprise antibodies, e.g., antibodiesthat bind specifically to the protein or peptide.

In some embodiments, the method further comprises sorting the cellsbased on expression of the protein or peptide. In some cases, whereinsorting the cells comprises using a Fluorescence Activated Cell Sorter(FACS).

In some embodiments, dividing the sample into subsamples comprisescombining fluids from multiple inlet channels. In some cases, combiningthe fluids from multiple inlet channels comprises combining aqueoussample fluid from a first inlet channel with a hydrophobic carrierfluid, e.g., oil, from at least one second inlet channel.

In one aspect, systems for detecting expression of a specific protein orpeptide by individual cells of a population of cells include: a fluidcontrol device configured to divide a sample comprising the populationof cells into subsamples, such that each subsample contains at most oneof the population of cells and each subsample is encompassed ahydrophobic fluid; rolling circle amplification reagents for detectionof the presence of the specific protein or peptide; and a deviceoperable to detect the rolling circle amplification reagents inindividual subsamples. Embodiments can include one or more of thefollowing features.

In some embodiments, systems also include a cell sorting mechanismoperable to sort the subsamples based on measurement of an indicatorparameter in each subsample. In some cases, the cell sorting mechanismcomprises a fluorescence activated cell sorting mechanism.

In one aspect, a kit includes: a fluid control device configured todivide a sample comprising the population of cells into subsamples, suchthat each subsample contains at most one of the population of cells andeach subsample is encompassed a hydrophobic fluid; and rolling circleamplification reagents for detection of the presence of the specificprotein or peptide. Embodiments can include one or more of the followingfeatures.

In some embodiments, kits also include a device operable to detect therolling circle amplification reagents in individual subsamples. In somecases, kits also include a cell sorting mechanism operable to sort thesubsamples based on measurement of an indicator parameter in eachsubsample. For example, the cell sorting mechanism can include afluorescence activated cell sorting mechanism.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Methods and materials are described hereinfor use in the specific systems and methods; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

We have demonstrated real time RCA of cancer cell surface specificmarkers in a microfluidic format. A microfluidic system was used toencapsulate single cells in monodisperse pico-liter RCA reactiondroplets. Inclusion of fluorescent probes in the RCA reaction mixpermitted the amplification process to be monitored within individualdroplets. The resulting fluorescent signal can be the basis formicrofluidic cell sorting based on a cell surface marker.

A PDMS microfluidic device was used for the production of monodisperseaqueous emulsion droplets with each droplet containing at most a singlecell with a RCA reaction mix. This microfluidic system for RCA offersthe advantage of a reduction of reaction volumes, and thereby decreasesin reagent and sample consumption which can be critical when limitedanalyte material is available from clinical samples. Since RCA is anisothermal reaction there is no need for thermal cycling that requiresophisticated and expensive instrumentation if compared to microfluidicsystems for PCR (see e.g., Konry et al., Analytical Chemistry 81(14),5777-5782 (2009); Demidov, Expert Rev. Mol. Diagn., 2(6), 89-95, (2002);and Landegren et al., Comp. Funct. Genomics, 4, 525-530, (2003)).Additionally, the new technique will potentially, due to the largemultiplexing capacity of RCA, the methods described herein enableparallel detection of various markers thus providing a highly multiplexmethod by using different combinations of spectrally resolvablefluorophores. Such versatility is superior over PCR-based approaches,since multiplex PCR needs optimization for each primer pair used andstill has problems with uniformity.

Practical applications for the disclosed amplification labeling methodsand systems for cell surface specific markers include, for example, incancer cell diagnostics. The identification of cell surface antigens iscritical to the development of new diagnostic and therapeutic modalitiesfor the management of cancer. EpCAM, or ‘Epithelial Cell AdhesionMolecule’ is a pan-epithelial differentiation antigen that is expressedon almost all carcinomas. Therefore detection of EpCAM-expressing cellsin the sample serves as the first screening test for cancer. Inaddition, this specific tumor cell marker is one criterion used incirculating tumor cell (CTC) identification. Usually, anti-EpCAMantibody is used to identify the CTCs. Cells that express this specificcancer marker can serve as molecular targets for sensitive and specificdetection. However, tumor cells that express low levels of EpCAM may notdetected directly by antiEpCAM antibodies due to low signal intensity.Therefore, the described systems and methods can provide the ability todetect differential expression of EpCAM on the cancer cell using RCAsignal that can reduce the likelihood that any target cells are missed.

The PDMS microfluidic devices can be used for production of monodisperseaqueous emulsion droplets of picoliter or nanoliter in size in acontinuous oil phase to encapsulate each cell in their ownmicroenvironment. In this hydrodynamic system, focusing, the size of aliquid jet can be considerably reduced to achieve faster mixing time.Since the volume of each drop is restricted, molecules secreted by anindividual cell can rapidly attain detectable concentrations. Eachdroplet contains up to one individual cell encapsulated along withdetection reagents, such as fluorescently labeled detection antibodiesand conjugated microshperes for secreted analyte measurement (dependingon the number of cells, some droplets may have no cells, but optimallyno droplets will have more than one cell). In some embodiments, secretedanalytes bind to microspheres previously conjugated withanalyte-specific antibodies and this binding is detected in real timevia detection of a fluorescent signal from labeled antibodies alsopresent in the droplets. In the case of no analyte secretion from thecell, the fluorescence diffuses in the droplet and is not localized onthe microsphere surface. Because the cells in the droplets remain viableafter encapsulation, the cells can be captured based on signalsdetected, and cultured to produce clonal populations of cells, e.g.,using known cell-sorting and culture methods. It is also possible thatthe same set of droplets could be used overtime to produce multiplecopies of single cell droplets-array at different time points. Thismethod has an advantage in the fabrication process and device handling,which avoids washing and surface modification steps as compared topreviously published single cell secretion analysis. In addition, othermethods for the analysis of individual cells in large numbers do notallow both high-throughput analysis of a secreted product and recoveryof living cells for clonal expansion.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figure, and from the claims.

DESCRIPTION OF DRAWING

FIG. 1 is a schematic illustration of an RCA assay for the detection ofa target surface protein.

FIG. 2 is a schematic illustration of a microfluidic cell sorting systemfor RCA in drops.

FIGS. 3A to 3D are a series of microscope images of PC3 cells fixed on aglass slide.

FIG. 4 is a schematic illustration of experimental setup.

FIGS. 5A to 5C are a series of microscope images of microfluidic devicesfor encapsulation in created droplets.

FIG. 5D is a photograph of an incubation and analysis channel.

FIGS. 6A and 6B are a series of (A) bright field and (B) fluorescencemicroscope images of aqueous droplets containing T cells, beads, andfluorescent secondary antibody.

DETAILED DESCRIPTION

The systems and methods described herein are based, at least in part, onthe discovery of methods for detecting molecules secreted by singlecells and rare cell surface markers. In some embodiments, the methodsuse rolling circle amplification with microfluidic devices forencapsulation, incubation, and analysis of cell surface markers orsecreted molecules from a single cell. Detection of cell surfacespecific markers in microfluidic format can be utilized in a number ofapplications, e.g., in cancer cell diagnostics.

Cell Surface Markers

A cell surface marker is a molecule found on the external cell wall orplasma membrane of a specific cell type or a limited number of celltypes (see e.g., Molday and Maher, Histochemical Journal 12:273-315(1980); Hewett, International Journal of Biochemistry & Cell Biology33:325-335 (2001); and Pembrey et al., Applied and EnvironmentalMicrobiology 65:2877-2894 (1999)). These markers are of importance inclinical studies and development of new diagnostic and therapeuticmodalities for the management of human diseases such as cancer (seee.g., Molday and Maher, Histochemical Journal 12:273-315 (1980); Hewett,International Journal of Biochemistry & Cell Biology 33:325-335 (2001);and Pembrey et al., Applied and Environmental Microbiology 65:2877-2894(1999)). Various therapeutic strategies could be employed to exploit theselective expression of targets on the surface of tumor-associated cellsuch as antibody- or gene-directed therapy, and small moleculeapproaches (Hewett, International Journal of Biochemistry & Cell Biology33:325-335 (2001)). These therapeutic approaches depend on theidentification of suitable selective targets on the associated tumorcell (Hewett, International Journal of Biochemistry & Cell Biology33:325-335 (2001)). A significant challenge in diagnostic analysis ofthese targets via immunolabeling is insufficient or low-intensitydetection signal due to low expression of the target. This leads toinefficient use of existing antibodies and a need for sensitive signalamplification technique. Therefore, the present methods, which caninclude use of the RCA technique can be used to enhance the intensity ofthe signal of the interrogated cell surface marker.

FIG. 1 schematically illustrates an exemplary RCA assay for thedetection of analytes, e.g., a target surface protein. In this example,anti-EPCAM antibody binds to a target EpCAM protein and is in turn boundby secondary biotin-labeled antibodies (step A). An avidin bridgecaptures a biotinylated DNA probe (step B). Cyclic DNA is hybridized tothe primer (step C), and the capture probe is extended by polymerase(step D).

It will be understood by skilled practitioners that any molecule that issecreted or found on the cell wall or plasma membrane of any cell, e.g.,a marker can be detected. For example, cell surface markers that areexpressed on a specific cell type or a limited number of cell types canbe detected. The process will be described for use with cancer cells,although it may be adapted for use with other cells, e.g., stem cellsand immune cells. Examples of cell surface markers include, but are notlimited to, membrane proteins such as receptors, transporters, ionchannels, proton pumps, and G protein-coupled receptors; extracellularmatrix molecules such as adhesion molecules (e.g., integrins, cadherins,selectins, or NCAMS); See, e.g., U.S. Pat. No. 7,556,928, which isincorporated herein by reference in its entirety.

Diagnostic markers for T-cells include the T-cell receptor (TCR) withits associated CD3 signaling complex as well as CD5 and the E-receptor(CD2). The presence of membrane immunoglobulin (mIg), which functions asantigen receptor, is diagnostic for B-cells. Complement receptors (CR)and Fc receptors (FcR) which can mediate opsonization, and MHC Class IImolecules which are important in antigen presentation, are present onB-cells and macrophages, as well as dendritic cells. Two major classesof T-cells are distinguished by the presence of either CD4 (on T_(H) andT_(reg)) or CD8 (on T_(C)). T_(H) cells can be further subdivided intoT_(H1) and T_(H2), which produce different cytokines and have distinctphysiological roles.

Secreted Molecules

As used herein, secretion is the process of releasing a molecule from acell, and occurs in prokaryotes and eukaryotes. For example, secretionis an important mechanism in bacterial functioning and operation intheir natural surrounding environment for adaptation and survival.Eukaryotic cells have a highly evolved process of secretion, which mayinvolve the classical endoplasmic reticulum (ER)-Golgi pathway. Manymammalian cell types have the ability to be secretory cells and thushave a well developed ER and Golgi apparatus to fulfill this function.Cells in humans that produce secretions include those in thegastrointestinal tract, which secretes digestive enzymes and gastricacid; the lung, which secretes surfactants; sebaceous glands, whichsecrete sebum to lubricate the skin and hair; the pancreas, whichsecretes insulin; and meibomian glands in the eyelid, which secretesebum to lubricate and protect the eye. Further, cells of the immunesystem can secrete cytokines and proteins.

Secretome analysis can be used e.g., to examine molecules secreted fromspecific cells, measure time-dependent variability of secretion, andidentify unique types of cells that respond to activation with specificanalyte secretion. The process will be described for use with IL-10,although it may be adapted for use with other secreted molecules, e.g.,proteins, peptides, enzymes, cytokines, chemokines, hormones, toxins,and antimicrobial peptides.

To demonstrate the ability of droplet based single cell secretionanalysis, IL-10 secretion was measured from a CD4+CD25+ regulatory Tcell clone. IL-10 is an anti-inflammatory cytokine with pleiotropicactivities on 13, T, and mast cells and is produced by a variety of celltypes in response to activation (Pestka et al., Annu Rev Immunol.22:929-79 (2004)). It has been reported that diminished IL-10 productionis associated with autoimmunity (Astier and Haller, J Neuroimmunol.191:70-8 (2007)). Thus, since IL-10 can exert its inhibitory activitythrough multiple effects on different cell types, it is important tounderstand how its secretion is regulated and to examine the uniquecells that respond to IL-10. Understanding how IL-10 secretion isregulated in different cell types would be markedly enhanced by theability to detect and re-isolate only those viable cells that areactively secreting IL-10 from the majority of cells in the populationthat are not producing IL-10. The technology described herein can beused to isolate IL-10 secreting cells by encapsulating cells, and laterrecovering the cells from the drops (e.g., using FACS).

It will be understood by skilled practitioners that this method can alsobe applied to multitarget detection. Additional individually detectablemicrospheres that bind other targets can be incorporated in this type ofsecretion measurement. In this way, a single cell secretome can beachieved using multiple microspheres previously encoded with differentcapturing antibodies. The ability to use combinations of analytes forsignal generation should enables simplification of sorting and detectionanalysis. For example, since monitoring of CD4+CD25+ regulatory T cellsshows promise in cancer immunotherapy, the system can be applied tostudy new paradigms for designing cytokine antagonists and cell-cellregulation. The encapsulation of two different cells in the same dropletto study the direct interaction between them (with the secretion of acytokine or the upregulation of a cell surface receptor as output), andscreening of different blocking/agonistic antibodies to surfacereceptors could be some of the various further applications to thissystem.

FIG. 2 is a schematic illustration of a microfluidic cell sorting systemfor RCA in drops. The microfluidic device produces monodisperse aqueousemulsion droplets containing single cells and an RCA reaction mix. Thedroplets can then be incubated (e.g., stored in an incubation region ofthe microfluidic device).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: RCA Amplification of Epithelial Cell Adhesion Molecule(EpCAM) Using the PC3 Human Prostate Cancer Cell Line

EpCAM is a pan-epithelial differentiation antigen that is expressed onalmost all carcinomas, therefore detection of EpCAM-expressing cells ina sample can serve as first screening test for cancer (Spizzo et al.,Breast Cancer Research and Treatment 86:207-213 (2004)). In addition,this specific tumor cell marker is usually used to identify acirculating tumor cell (CTC) (Nagrath et al., Nature 450:1235-1238(2007)). However tumor cells that express a low level of EpCAM may notbe detected directly by anti-EpCAM antibodies due to low signalintensity (Deng et al., Breast Cancer Research 10:1-11 (2008)).Therefore, the ability to detect differential expression of EpCAM on thecell using a RCA signal can ensure that no target cells are missed.

An RCA signal can be confined locally if the amplification primer isimmobilized onto a solid support (Konry et al., Analytical Chemistry 81:5777-5782 (2009); Smolina et al., Applied and Environmental Microbiology73:2324-2328 (2007)) and the resulting signal can be detected directlyvia fluorescent microscopy and/or digitally quantified with a GenePix®microarray scanner (Smolina et al., Applied and EnvironmentalMicrobiology 73:2324-2328 (2007)). Two sets of experiments wereperformed. Representative results obtained in these experiments arepresented in FIGS. 3A-3D.

Microscope images of PC3 cells fixed on a glass slide previously labeledwith DAPI (blue) were taken. The PC3 cells were labeled directly withbiotinylated anti-EpCAM antibodies and Cy3 fluorescent labeledstreptavidin. The specificity of the system was also tested by applyingthe same conditions to lymphocytes. As lymphocyte cells do not expressEpCAM, no fluorescent signal was detected using the direct detectionmethod.

In this experiment, PC3 cells were labeled using the RCA technique. Inthis approach, the cells were first labeled with biotinylated anti-EpCAMantibodies. Biotinylated primers were immobilized via a biotin-avidinbridge to the biotinylated anti-EpCAM antibodies. Next, circulartemplate (padlock) was immobilized to the primers and isothermallyamplified by phi29 DNA polymerase. As the amplification is isothermal,the RCA process preserves the integrity of antibody-antigen (EpCAM)complexes. The RCA product contains thousands of repeat sequences thatcontain fluorescently labeled nucleotides. RCA amplifies the detectionsignal and allows for the detection of PC3 cancer cells.

Isothermal amplification using phi29 DNA polymerase was also performedin droplets. In order to evaluate the method for screening asingle-cell, a drop-based PDMS microfluidic device was applied toencapsulate PC3 cells in distinct picoliter-sized drops of RCA reagents.A PDMS microfluidic system which generates monodisperse droplets in amicrochannel through shearing flow at a T-junction or a flow-focusingzone has been previously described (Kiss et al., Anal. Chem.80:8975-8981 (2008); Köster et al., Lab on a Chip, 8:1110-1115 (2008)).Other methods of generating droplets (e.g., separating the sample intosubsamples) could also be used.

The RCA reaction droplets were conveyed by the oil flow through channelsin a microfluidic chip. Individual droplets were then focused in thearray channel for amplification reaction of cell surface marker andoptical interrogation (FIGS. 3A to 3D). An array like channel permitsincubation for polymerization and measuring of fluorescence signal inreal time from each individual cell, providing information onamplification efficiency within each droplet. The array channel hereinpermits simultaneous interrogation of multiple droplets/reactions/cellsand it can be easily designed to hold thousands of droplets (Kiss etal., Anal. Chem. 80:8975-8981 (2008)). Comparing the fluorescent imageof an encapsulated cell in an RCA reaction at the beginning of theincubation (see FIG. 3C) with the cell after 1.5 hours (see FIG. 3D), itis clear that the RCA was performed on a single PC3 cancer cell enclosedin a droplet. In this research setting, real-time RCA in pL-drops canprovide quantitative measurements, if fluorescence was recorded using acustom optics system and software similar to one described by Kiss etal. (Anal. Chem. 80:8975-8981 (2008)).

The basic cell encapsulation device uses a flow focusing geometry toproduce droplets (Brouzesa et al., Proc. Natl Acad. Sci. USA 106:14195-14200 (2009); Edd et al., Lab Chip 9:1859-65 (2009); Köster etal., Lab Chip 8:1110-1115 (2008); Wan et al.) FIG. 4 illustrates a PDMSdroplet-based microfluidic device. Three inlet channels form a nozzle astheir flows combine (see FIGS. 5A and 5B). The center stream containsbeads, cell and secondary antibodies suspension while the side streamscontain the oil phase. The size of the droplets controlled by matchingthe size of the nozzle orifice to the drop diameter and operating thedevice in the dripping regime. For drop formation, the flow rate ratioof water to oil was adjusted to the Qw/Qo=0.5. For single cell studies,all drops should optimally contain at most one cell, so that themajority of drops contain no cell at all since the encapsulation processfollows Poisson statistics. Although the number of single-cell-bearingdrops is rather low, for these experiments this is not severe, given thehigh production and screening rate that can be achieved withmicrofluidic devices.

Microfluidic Device Fabrication

Microfluidic flow chambers were fabricated by soft lithography. Negativephoto resist SU-8 2025 or SU-8 2100 (MicroChem, Newton, Mass.) wasdeposited onto clean silicon wafers to a thickness of 50 lm, andpatterned by exposure to UV light through a transparency photomask(CAD/Art Services, Bandon, Oreg.). The Sylgard 184poly(dimethylsiloxane) (PDMS) (Dow Corning, Midland, Mich.) was mixedwith crosslinker (ratio 10:1), poured onto the photoresist patterns,degassed thoroughly and cured for at least 1 hour at 65° C. The PDMSdevices were peeled off the wafer and bonded to glass slides afteroxygen-plasma activation of both surfaces. To improve the wetting of thechannels with mineral oil, the microfluidic channels are treated priorto the experiments with Aquapel (PPG Industries, Pittsburgh, Pa.) byfilling the channels with the solution as received and then flushingthem with air. Polyethylene tubing with an inner diameter of 0.38 mm andan outer diameter of 1.09 mm (Becton Dickinson, Franklin Lakes, N.J.)connected the channels to the syringes. Syringes were used to load thefluids into the devices, while the flow rates were controlled by syringepumps. Optimum devices for drop formation and cell encapsulation are 40lm high with a 35 lm-wide nozzle. To vary the drop size, we also used achannel height of 25 lm and different nozzle widths. The channel forcell incubation are 100 lm high, the 1 width is 500 lm, and the lengthis 2.88 m. All inlet channels were equipped with patterned filters whichprevent dust particles from clogging the channels downstream.

As lymphocyte cells do not express EpCAM, no fluorescent signal wasdetected using the RCA methods.

CD4+CD25+ High Regulatory T Cell Cloning

Whole mononuclear cells were isolated from human blood drawn fromhealthy control donors by Ficoll-Hypaque (Amersham Biosciences) gradientcentrifugation, and total CD4 T cells were isolated by negativeselection using a CD4+ T-cell isolation kit II (Miltenyi Biotec,Bergisch Gladbach, Germany) and stained for fluorescence-activated cellsorting (FACS) with antibodies against CD45RA (HI100), CD25 (M-A251),and HLA-DR (L-243). The specific DR−Treg (CD45RA-CD25highDR−)(Baecher-Allan et al., J Immunol. 176:4622-31 (2006)), and memory Tresponder (CD45RA-CD25med) populations were sorted in a FACSAria™ cellsorter (BD Biosciences) at one cell per well in XVIVO-15 (Lonza) mediumcontaining 5% human serum and stimulated with soluble anti-CD3 (cloneHit3a, BD Biosciences) and anti-CD28 (clone 28.2) (both at 1 μg/mL),irradiated APCs (105/well), and IL-2 (50 U/mL). Half of the medium wasreplaced with fresh medium containing IL-2 (50 U/mL) starting at day 10and every 3 to 4 days thereafter. After 4 weeks of expansion, each clonewas tested for FoxP3 expression and IL-10 production.

Preparation of Microsphere Sensors

SPHERO™ Avidin Coated Particles (0.7-0.9 μm) were conjugated withbiotinylated IL-10 monoclonal capture antibodies (Invitrogen AHC7109)and suspended in PBS. A microcentrifuge tube containing the mixture wasshaken at 25° C. for 4 hours. The microspheres were washed once with 300μL Tris-Starting Block (blocking buffer) and suspended in 300 μLblocking buffer. The suspension was shaken at 25° C. for 30 minutes andthen washed once with 300 μL blocking buffer. The microsphere probeswere suspended and stored in 100 μL blocking buffer at 4° C.

A mixed solution of cells, anti-IL-10 conjugated microspheres (1 mg),and rat anti-human IL-10 FITC conjugated antibodies, at a concentrationof 1 μg/mL (Invitrogen RHCIL1001) were ejected for encapsulation with asyringe. After encapsulation, the flow in the channels was stopped andthe outlet of the device was blocked allowing previously encapsulateddroplets to be incubated for 3 hours.

Image Analysis

Fluorescence images were captured on a Zeiss 200 Axiovert microscopeusing an AxioCAM MRm digital camera. FITC fluorescence (excitation 488nm, emission 525 nm) was monitored to evaluate the microsphere assay(FIG. 6B).

The method described combines the sensitivity of enhanced RCA detectionwith a reduction in reaction volumes, reagent, and sample consumptiondue to the microfluidic format. This system provides high-throughput,specific marker amplification of thousands of reactions per hourcombined with real-time monitoring of individual reactions. In addition,various therapeutic strategies such as antibody- or gene-directedtherapy, and small molecule approaches, exploiting the selectiveexpression of targets on the surface of tumor-associated cell, couldbenefit from this sensitive and rapid protein surface detection method.The correlations between surface protein expressions data obtained froma tumor cell before treatment and after the responses of patients tovarious therapeutic regimens can be obtained on a single cell level. Inaddition, individual tumor cell analysis, if applied for detection ofCTCs, could provide important information, once a cell is recovered froma drop for future testing.

Example 2: Microfluidic Cell Sorting Based on IL-10 Secretion

Single CD4+CD25+ regulatory T cells were encapsulated in distinctnL-sized microenvironment drops and IL-10 secretion was measured in theincubation channel (FIGS. 5D, 6A, and 6B). A detectable concentration ofIL-10 secreted by an individual cell was reached in 2 to 3 hours in therestricted volume of droplet (FIGS. 6A and 6B). The droplets with asingle cell that produced IL-10 were detected via a fluorescent signalaccumulated on the surface of microspheres. FIGS. 6A and 6B depictaqueous droplets containing T cells, beads, and fluorescent secondaryantibody after 3 hours incubation in the channel. The microspheres andcells can be seen in the bright field image (FIG. 6A). FIG. 6B (leftpanel) shows the fluorescence image where secondary fluorescentlylabeled antibody has been localized on the beads, indicating thesecretion of IL-10 from the cell and later binding to the microspheres.FIGS. 6A and 6B (right panels) present an encapsulated T cell which isnot secreting IL-10 from the same batch experiment.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-8. (canceled)
 9. A system for detecting expression of a specificprotein or peptide by individual cells of a population of cells, thesystem comprising: a fluid control device configured to divide a samplecomprising the population of cells into subsamples, such that eachsubsample contains at most one of the population of cells and eachsubsample is encompassed a hydrophobic fluid; rolling circleamplification reagents for detection of the presence of the specificprotein or peptide; and a device operable to detect the rolling circleamplification reagents in individual subsamples.
 10. The system of claim9, wherein the rolling circle amplification reagents comprise a phi29DNA polymerase or a circular DNA template or both.
 11. The system ofclaim 9, wherein the rolling circle amplification reagents comprise abiotinylated antibody that binds specifically to the protein or peptide,an avidin bridge, a biotinylated DNA probe, and a circular DNA templatecapable of hybridizing with the biotinylated DNA probe.
 12. The systemof claim 11, wherein the rolling circle amplification reagents furthercomprise a phi29 polymerase and a fluorescently-labeled nucleotide. 13.The system of claim 11, wherein the biotinylated antibody bindsspecifically to a protein or peptide expressed by a cancer cell, a stemcell, or an immune cell.
 14. The system of claim 11, wherein thebiotinylated antibody binds specifically to an epithelial cell adhesionmolecule (EpCAM).
 15. The system of claim 9, further comprising a cellsorting mechanism operable to sort the subsamples based on measurementof an indicator parameter in each subsample.
 16. The system of claim 15,wherein the cell sorting mechanism comprises a fluorescence activatedcell sorting mechanism.
 17. The system of claim 9, wherein dividing thesample into subsamples comprises combining aqueous sample fluid from afirst inlet channel with a hydrophobic carrier fluid.
 18. A kitcomprising: a fluid control device configured to divide a samplecomprising the population of cells into subsamples, such that eachsubsample contains at most one of the population of cells and eachsubsample is encompassed a hydrophobic fluid; and rolling circleamplification reagents for detection of the presence of the specificprotein or peptide.
 19. The kit of claim 18, wherein the rolling circleamplification reagents comprise a phi29 DNA polymerase or a circular DNAtemplate or both.
 20. The kit of claim 18, wherein the rolling circleamplification reagents comprise a biotinylated antibody that bindsspecifically to the protein or peptide, an avidin bridge, a biotinylatedDNA probe, and a circular DNA template capable of hybridizing with thebiotinylated DNA probe.
 21. The kit of claim 20, wherein the rollingcircle amplification reagents further comprise a phi29 polymerase and afluorescently-labeled nucleotide.
 22. The kit of claim 20, wherein thebiotinylated antibody binds specifically to a protein or peptideexpressed by a cancer cell, a stem cell, or an immune cell.
 23. The kitof claim 20, wherein the biotinylated antibody binds specifically to anepithelial cell adhesion molecule (EpCAM).